Crystallization and characterization of human liver kynurenine--glyoxylate aminotransferase. Identity with alanine--glyoxylate aminotransferase and serine--pyruvate aminotransferase.
Kynurenine-glyoxylate aminotransferase, alanine-glyoxylate aminotransferase and serine-pyruvate aminotransferase were co-purified and crystallized as yellow cubes from human liver particulate fraction. The crystalline enzyme was homogeneous by the criteria of electrophoresis, isoelectric focusing, gel filtration, sucrose-density-gradient centrifugation and analytical ultracentrifugation. The molecular weight of the enzyme was calculated as approx. 90000, 89000 and 99000 by the use of gel filtration, analytical ultracentrifugation and sucrose-density-gradient centrifugation respectively, with two identical subunits. The enzyme has a s(20,w) value of 5.23S, an isoelectric point of 8.3 and a pH optimum between 9.0 and 9.5. The enzyme solution showed absorption maxima at 280 and 420nm. The enzyme catalysed transamination between several l-amino acids and pyruvate or glyoxylate. The order of effectiveness of amino acids was alanine>serine>glutamine>glutamate>methionine>kynurenine = phenylalanine = asparagine>valine>histidine>lysine>leucine>isoleucine>arginine>tyrosine = threonine>aspartate, with glyoxylate as amino acceptor. The enzyme was active with glyoxylate, oxaloacetate, hydroxypyruvate, pyruvate, 4-methylthio-2-oxobutyrate and 2-oxobutyrate, but showed little activity with phenylpyruvate, 2-oxoglutarate and 2-oxoadipate, with kynurenine as amino donor. Kynurenine-glyoxylate aminotransferase activity was competitively inhibited by the addition of l-alanine or l-serine. From these results we conclude that kynurenine-glyoxylate aminotransferase, alanine-glyoxylate aminotransferase and serine-pyruvate aminotransferase activities of human liver are catalysed by a single protein. Kinetic parameters for the kynurenine-glyoxylate aminotransferase, alanine-glyoxylate aminotransferase, serine-pyruvate aminotransferase and alanine-hydroxypyruvate aminotransferase reactions of the enzyme are presented.
D000368:Aged; D000410:Alanine Transaminase / Q000378:metabolism*; D003460:Crystallization; D006801:Humans; D007700:Kinetics; D007737:Kynurenine / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D012694:Serine / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D000637:Transaminases / Q000378:metabolism*
D007737:Kynurenine; D012694:Serine; D000637:Transaminases; D000410:Alanine Transaminase; C019635:Alanine-glyoxylate transaminase; C022638:serine-pyruvate aminotransferase